Discovery of Novel Synthetic Cyclohexene-Based Small Molecules Targeting Senescence against Age-Related Pulmonary Fibrosis

Abstract

Targeting cellular senescence has emerged as a therapeutic strategy for the increasingly prevalent age-related diseases, yet no drugs have reached clinical approval specifically as senotherapeutics. Through senescence-phenotype screening of our recently generated human microbiota-inspired library of small molecules, we identified a tetrasubstituted cyclohexene as a novel bioactive chemotype. Following the synthesis of related analogues and their evaluation in relevant models of senescence, we discovered compound 25 (UCM-17017) that decreases β-galactosidase activity in senescent human fibroblasts and selectively reduces the viability of senescent human lung adenocarcinoma cells over proliferative cells. The new senolytic compound exhibits a favorable pharmacokinetic profile in vivo and induces a beneficial effect in a mouse model of pulmonary fibrosis, a senescence-related disease with significant unmet medical needs. Our results are valuable for senotherapeutic drug discovery and support the interest of targeting cellular senescence as a promising approach against age-related pulmonary fibrosis.

Introduction

In recent decades, the considerable increase in life expectancy worldwide has led to a higher prevalence of chronic diseases associated with aging. According to the World Health Organization, these so-called age-related diseases, are among the leading causes of disability and morbidity in the elderly population. , Indeed, the “geroscience hypothesis” considers aging to be the main risk factor for most serious chronic pathologiesincluding cancer, neurodegenerative processes, chronic pulmonary diseases, cardiovascular diseases, atherosclerosis, diabetes, osteoporosis, osteoarthritis, hepatic dysfunction, renal failure, and blindness. − Thus, addressing an intervention that can slow down the aging process could reduce or postpone the incidence of debilitating age-related diseases, which should significantly reduce the enormous social and economic burden caused by these chronic diseases. , Among the accepted hallmarks of aging, cellular senescence plays a key role; − it is characterized by a stable cell-cycle arrest, deregulated metabolism, macromolecular damage, resistance to cell death and secretion of proinflammatory substances (cytokines, chemokines, extracellular matrix remodeling enzymes and growth factors, among others), collectively known as senescence-associated secretory phenotype (SASP).

Senescence is an essential physiological program that occurs in normal cells in response to cellular stress, having beneficial and protective effects for the organism. These effects include tumor suppression by limiting the proliferation of damaged or unwanted cells, as well as limiting the occurrence of fibrosis by favoring tissue repair. , However, the accumulation of senescent cells throughout life, mostly due to a weakened immune system, causes numerous adverse effects and contributes to the development of age-related pathologies. , Therefore, targeting cellular senescence has emerged as a therapeutic strategy to prevent and mitigate age-related diseases and to increase lifespan and healthspandefined as total years of life lived in good health and without disability. ,,, In this regard, compounds with antisenescence potentialglobally known as senotherapeuticshave been identified as molecules capable of eliminating senescent cellssenolyticsor suppressing the senescence phenotype responsible for inflammation and impaired tissue regenerationsenomorphics. The anticancer drug dasatinib (D, Figure ) and the natural flavonoid quercetin (Q, Figure ) were the first senotherapeutics described in 2015, exhibiting senolytic efficacy in mice. The combination treatment D+Q has demonstrated efficacy in mouse models of atherosclerosis, pulmonary fibrosis, hepatic steatosis, Alzheimer’s disease and obesity, among others. Since then, a number of senolytics have been reported, mainly based on natural products, such as the flavonoid fisetin (Figure ), or from the repurposing of anticancer drugs, such as navitoclax (ABT-263, Figure ), inhibitor of the BCL-2 family of antiapoptotic proteins, and cardiac glycosides. , The senolytic combination D+Q has entered several clinical trials for the treatment of Alzheimer’s disease, idiopathic pulmonary fibrosis, chronic kidney disease, frailty, and age-related bone loss. The human efficacy of the flavonoid fisetin in age-related diseases is also currently being evaluated in several clinical trials for frail elderly syndrome and knee osteoarthritis therapies. Among senomorphic compounds, rapamycin (Figure ), indicated for the prophylaxis of organ transplant rejection, and metformin (Figure ), a synthetic drug approved for the treatment of type 2 diabetes, have been shown to reduce the accumulation of β-amyloid and tau proteins and improve cognition in animal models. − Clinical trials of senomorphic rapamycin for early Alzheimer’s disease and amyotrophic lateral sclerosis are presently underway.

1.

Structures of the most studied senotherapeutic compounds.

Hence, targeting cellular senescence has clearly emerged as a novel strategy to address different age-related pathologies and numerous preclinical studies support the potential of senotherapeutics. However, trials aimed at confirming their clinical indication and long-term safety are progressing slowly, and there are currently no drugs approved specifically as senotherapeutics. ,

In the present work, we contribute to the desired search for novel compounds with senotherapeutic activity using an in-house chemical library that includes our recently generated focused library of small molecules inspired on human microbiota metabolites. Following the identification of tetrasubstituted cyclohexene scaffold as a new bioactive chemotype, the synthesis of related analogues and their evaluation in relevant cellular senescence phenotypes, we discovered compound 25 (UCM-17017) that leads to a decrease in the activity of senescence-associated β-galactosidase (SA-β-gal), reduces viability of senescent human lung adenocarcinoma cells with specificity over proliferative cells, and exhibits a favorable pharmacokinetic profile in vivo. The new senolytic agent induces a beneficial effect in a mouse model of pulmonary fibrosis, one of the most common diseases directly related to the senescence process and with unmet medical needs. Our results are valuable for senotherapeutic drug discovery that could provide new therapies against age-related pulmonary fibrosis.

Results and Discussion

Identification of New Senotherapeutic Compounds

The lack of a single and well-established senescence marker makes it difficult to identify a potent, nontoxic and selective senotherapeutic drug. For our primary screening, we have used the enzyme β-galactosidase that is overexpressed in senescent cells and is one of the most widely used biomarkers for senescence detection. The senescence phenotype was generated using human fibroblasts from lung tissue (IMR-90) that were treated with H₂O₂ as oxidative stress. The activity of β-galactosidase in senescent cells was determined by measuring fluorescein emission at 520 nm from the hydrolysis of fluorescein-di-β-galactopyranoside (FDG), and this fluorimetric SA-β-gal assay was used as screening for the identification of compounds affecting cellular senescence.

An in-house chemical library of 256 compounds was first tested at 10 μM and the measured fluorescence intensity indicated β-galactosidase activity related to the senescence phenotype of the cells. Compounds that reduced SA-β-gal activity to less than 55% were subsequently tested at 1 μM. Upon analysis of the screening results, we identified compounds 1 and S1–S9 able to reduce SA-β-gal activity to a value below 55% also at 1 μM (see Table S1). Among them, analogue 1 (Figure ) exhibited the lowest SA-β-gal activity both at 1 and 10 μM (44% and 16%, respectively). Next, compound 1 was evaluated in nonsenescent IMR-90 cells using the colorimetric MTT assay, resulting in a cell viability of ≈100% that indicates it is not cytotoxic. Considering the results obtained in the FDG SA-β-gal and MTT assays, compound 1 was selected as a new chemotype in the search for synthetic senotherapeutic compounds. Interestingly, 1 was identified within our recently generated set of small molecules inspired on human microbiota metabolites. Indeed, compound 1 contains a central tetrasubstituted cyclohexene core related to shikimic acid, a metabolite of the human microbiota whose biological relevance has been highlighted in a recent study. Shikimic acid was shown to inhibit the proliferation and migration of vascular smooth muscle cells, suggesting a potential protective role in vascular wall diseases such as atherosclerosis and hypertension. This result provides further evidence for the influence of microbiota metabolites on human health and disease, particularly in age-related pathologies, an area of intense current research. −

2.

Search of new senotherapeutic compounds related to identified tetrasubstituted cyclohexene derivative 1 and discovery of the new senolytic compound UCM-17017.

For the synthesis of compound 1, the tetrasubstituted cyclohexene scaffold was built using an asymmetric aminocatalytic cascade (Scheme ). This multicomponent reaction affords with high enantio- and diastereoselectivity a chemotype characterized by the presence of three chiral carbon centers, also addressing the demand for compounds with an increased number of sp³ carbon atoms, a parameter that has achieved great significance in guiding drug research in recent years. , Altogether, the identification of 1 as a bioactive molecule provides rationale to our valuable methodology based on the design of new chemotypes containing privileged scaffolds present in microbiota metabolites and their obtention following synthetic routes based on asymmetric organocatalytic reactions.

1. Synthesis of Compounds 1–12 .

^a Reagents and conditions: (a) 20 mol % (R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine), toluene, 0 °C, 1 h, rt, on, 50%; (b) i. (cyclopropylmethyl)­amine, MeOH, rt, 3 h; ii. NaBH₄, rt, 3 h, 57%; (c) Zn, AcOH/MeOH, rt, 1–3 h, 80–90%; (d) valeric or nicotinic acid, EDC, HOBt, DIPEA, DCM, 0 °C to rt, 3 h, 18–36%; (e) Fmoc-OSu, DCM, rt, 3 h, 64%; (f) Boc₂O, Et₃N, DCM, rt, 3 h, 75%; (g) i. R-COOH, EDC, HOBt, DIPEA, DCM, 0 °C to rt, 3 h; ii. piperidine, DCM, rt, 8–16 h, 14–22% (for two steps); (h) HSiCl₃, DIPEA, DCM, rt, on, 74%; (i) glutaric anhydride, DMF, rt, on, 35%; (j) methyl 5-bromovalerate, K₂CO₃, KI, ACN, rt, 4 h, 35%; (k) LiOH·H₂O, THF:H₂O, rt, 48 h, 62%; (l) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, Et₃N, ACN, rt, on, 30%; (m) N _α-Boc-L-tryptophan, EDC, HOBt, DIPEA, DCM, rt, 3 h, 34%; (n) HCl (4 M in dioxane), rt, on, 49–75%.

Building upon the tetrasubstituted cyclohexene scaffold of compound 1, new analogues 2–33 were synthesized in the search for novel senotherapeutic agents, incorporating structural modifications at the primary amino group (R¹), the methyl substituent (R²) and the p-methoxyphenyl moiety (R³) (Figure and Schemes – ).

3. Synthesis of Compounds 18–33 .

^a Reagents and conditions: (a) 20 mol % (R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine), toluene, 0 °C, 1 h, rt, on, 19–75%; (b) i. (cyclopropylmethyl)­amine, MeOH, rt, 3 h; ii. NaBH₄, rt, 3 h, 20–94%; (c) Zn, AcOH/MeOH, 2 h, 16–81%; (d) BBr₃, DCM, 0 °C, 2 h, 65%.

Modifications around the primary amino group of 1 (R¹, Figure ) were explored in compounds 2–12 (Scheme ), by introduction of a selection of structural fragments present in numerous microbiota metabolites (short-chain fatty acids; benzoic, nicotinic, isonicotinic, and pipecolic acyl moieties; guanidine group; and tryptophan system). This set of compounds was synthesized following the synthetic route employed for compound 1, featuring an asymmetric three-component organocatalytic cascade as the key step (Scheme ). Hence, the reaction between propionaldehyde, acrolein and trans-p-methoxy-β-nitrostyrene catalyzed by the R enantiomer of the Jørgensen–Hayashi catalyst [(R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine)] provided the tetrasubstituted cyclohexene scaffold 34 as the major diastereoisomer (diastereoisomeric ratio, dr = 9:1, determined by ¹H-nuclear magnetic resonance (NMR) analysis of the reaction crude), which was isolated after chromatographic purification. The relative configuration of this intermediate was determined by ¹H NMR nuclear Overhauser effect (NOE) experiments and agrees with that reported for the asymmetric cascade. The absolute configuration was assigned according to the stereochemical outcome of the reported reaction. Cyclohexenecarbaldehyde 34 was then transformed into amine 35 by reductive amination reaction with (cyclopropylmethyl)­amine (Scheme ), observing the complete inversion at the chiral carbon bearing the nitro group, which results in a favorable relative trans configuration of this group and the aryl ring. Next, reduction of nitro group yielded compound 1 (Scheme ).

For analogues 2 and 3, incorporating a valeric acid aliphatic chain and a nicotinic acyl moiety, respectively, a direct coupling reaction of 1 with the corresponding carboxylic acid was performed under standard EDC (1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide) conditions, yielding target amides 2 and 3. However, both compounds were obtained in very low yield due to competing reactivity between the two amino groups present in 1 (Scheme ). To prevent undesired acylation in the synthesis of final compounds 4–7, the secondary amine of intermediate 35 was protected using an N-fluorenylmethoxycarbonyl (Fmoc) group. The resulting N-Fmoc derivative 36 was then reduced to amine 37, which was coupled with the corresponding carboxylic acid followed by deprotection to afford target compounds 4–7 (Scheme ). The yield of the amidation reaction under these conditions remained low, primarily due to partial in situ deprotection of 37, which hindered regioselective acylation. To address this limitation for the synthesis of the remaining analogues, the tert-butoxycarbonyl (Boc) group was explored as an alternative protecting group for intermediate 35 (Scheme ). In this approach, the nitro group of N-Boc protected derivative 38 was reduced using trichlorosilane and N,N-diisopropylethylamine (DIPEA), affording amine 39 that was then used as a common intermediate for the synthesis of final compounds 8–12. Ring-opening reaction of glutaric anhydride with 39 yielded derivative 40, which, upon treatment with hydrochloric acid, led to final compound 8. For analogues 9 and 10, the valeric acid moiety was introduced via alkylation of 39 with methyl 5-bromovalerate, followed by N-Boc deprotection of resulting intermediate 41, to afford final compound 9. Alternatively, hydrolysis of the ester group in 41 and subsequent acid treatment yielded compound 10. Finally, derivatives 11 and 12 were synthesized by reaction of 39 with a guanidine precursor and N-Boc protected tryptophan, respectively, followed by Boc deprotection.

In compounds 13–17 we explored the introduction of microbiota privileged scaffolds such as indole, thiazole, benzothiazole, pyridine, or morpholine, as R² modifications at the methyl group in 1 (Figure and Scheme ). Cyclohexenecarbaldehyde precursors 45–49 were synthesized by applying a modified version of the cascade reaction previously used for compound 34, in which a nucleophilic component bearing the corresponding R² group is added. , Specifically, a multicomponent cascade between acrolein, trans-p-methoxy-β-nitrostyrene, and indole or an appropriate R²-functionalized alcohol acting as nucleophile [(1,3-thiazol-2-yl)­methanol, (1,3-benzothiazol-2-yl)­methanol, (pyridin-2-yl)­methanol, and 2-(morpholin-4-yl)­ethan-1-ol] was carried out, using the Jørgensen–Hayashi catalyst, to yield compounds 45–49 (Scheme ). The experimental conditions of the cascade reaction were optimized to ensure the successful formation of the target intermediates, requiring the slow addition of acrolein to minimize polymerization side reactions. Under these conditions, cyclohexenecarbaldehydes 45–49 were obtained with good diastereoselectivity (dr = 8:2–9:1, determined by ¹H NMR analysis of the reaction crude), and the major diastereoisomer was isolated after chromatographic purification. Subsequent reductive amination to introduce the cyclopropylmethyl fragment, followed by nitro group reduction in intermediates 50–54, afforded the final compounds 13–17 (Scheme ). In all cases, the reductive amination proceeded with complete inversion at the chiral center adjacent to the nitro group, leading to a favorable relative trans configuration of the p-methoxyphenyl group with respect to the other two substituents of the cyclohexene ring in derivatives 50–54.

2. Synthesis of Compounds 13–17 .

^a Reagents and conditions: (a) 20 mol % (R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine), 25 mol % benzoic acid, CHCl₃, rt, on, 28–54%; (b) i. (cyclopropylmethyl)­amine, MeOH, rt, 3 h; ii. NaBH₄, rt, 3 h, 30–87%; (c) Zn, AcOH/MeOH, rt, 2 h, 37–95%.

For the structural exploration around the aryl group of 1 (R³, Figure ), we considered phenyl rings differently substituted in ortho, meta and para positions (compounds 18–29, 33, Scheme ), as well as the replacement with aromatic heterocycles such as pyridine, thiophene, and 1,3-oxazole (compounds 30–32, Scheme ). The synthesis of these new analogues was approached from cyclohexenecarbaldehyde intermediates 55–69, obtained via an organocatalytic cascade involving acrolein, propionaldehyde and an appropriate nitroalkene containing the desired R³ groups (Scheme ). As expected, the cascade reactions took place with high diastereoselectivity (dr = 9:1), except for intermediate 67, which exhibited a lower dr (6:4), attributed to epimerization promoted by the basicity of the pyridine. Next, reductive amination followed by nitro group reduction, according to the methodology previously applied, afforded final compounds 18–32 (Scheme ). Analogue 33, featuring a hydroxy-substituted phenyl ring, was readily obtained by demethylation of compound 1, using boron tribromide at low temperature (Scheme ).

From the data obtained in the FDG SA-β-gal assay (Table ), compounds 3–6 and 12, containing aromatic systems in R¹, were able to reduce SA-β-gal activity to less than 55% and were subsequently tested at 1 μM. At low concentration, only 5 displayed similar senotherapeutic activity to that of parent compound 1. In addition, compound 5 displayed no cytotoxicity in nonsenescent IMR-90 cells, as determined using MTT assay (viability = 97%).

1. Senotherapeutic Activity of Compounds 1–33 in the FDG SA-β-gal Assay.

^aValues are the mean ± SEM of at least three independent experiments with triplicate determinations; nd = not determined.

New compounds 13–17, resulting from the structural variations at R² moiety, exhibited senotherapeutic activity, with SA-β-gal activity values below 55% at 10 μM except in the case of morpholine derivative 17 (Table ). Thiazole and pyridine analogues 14 and 16 were also active at 1 μM and displayed no toxicity in nonsenescent cells (viability >97%).

The modifications in R³ moiety afforded the most active compounds, since very low SA-β-gal activities were observed at 10 μM, as shown in Table for analogues 18–33. In general, the different substituents were tolerated at all three positions of the phenyl ring, while heterocycles were less favorable (SA-β-gal activity, 30: 48%, 31: 48%, 32: 49%). Compounds containing methyl, chloro, trifluoromethyl, and trifluoromethoxy substituents were able to reduce SA-β-gal activity to less than 10% (see analogues 21–24, 28 and 29). When tested at 1 μM, compounds 18–22, 25, 28, 29 and 33 maintained activity and no toxicity was observed in nonsenescent cells (viability >93%).

Senolytic Activity and Pharmacokinetics of Selected Compounds

Compounds 1, 5, 14, 16, 18–22, 25, 28, 29, 33, able to reduce SA-β-gal activity in human senescent fibroblasts to values lower than 55% at 1 μM and devoid of toxicity in nonsenescent cells, were selected for further evaluation of senotherapeutic effects. Their potential as senolytics was assessed in human lung adenocarcinoma (A549) cells treated with bleomycin, a well-known senescence-inducing chemotherapeutic drug. Cell viability by MTT assay was measured in bleomycin-induced senescence A549 cells for the selected compounds at different concentrations (1 and 10 μM) and time points (24, 48, and 72 h) (Figure A). From this initial screening, we considered as potential senolytic compounds those above the threshold (the average for the whole population of senescent A549 cells plus 2 standard deviations): 22 (1 μM, 48 h), 29 (10 μM, 48 h), 25 (10 μM, 72 h) and 16 (10 μM, 72 h). Interestingly, selectivity for bleomycin-induced senescent over proliferating A549 cells was observed for compound 25, with the best performance after 48–72 h post-treatment at both 1 and 10 μM (Figure B). The senolytic drug ABT-263 was used as positive control in our viability assays.

3.

Senolytic activity of selected compounds: viability in senescent and proliferative human lung adenocarcinoma (A549) cells, using an MTT assay. (A) Scatter plot representing the normalized senolytic activity obtained with each compound tested in senescent A549 cells. (B) Relative cell viability (%) of compound 25 tested in proliferative (Pro) and bleomycin-induced senescent (Bleo) A549 cells at 24 (left), 48 (middle), or 72 h (right). (C) Relative cell viability (%) of compounds 25, 85–89, 97, and 98, at 10 μM for 24 h. ABT-263 (ABT) was used as a positive control of senolysis. Statistical significance was assessed by the two-tailed Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001.

Based on these data, we synthesized new analogues 85–89 related to compound 25 to explore the replacement of the (cyclopropylmethyl)­amine moiety for amino, methylamino, acetamido, hydroxy, and methoxy groups (X-R⁴), while maintaining the primary amine (R¹), the methyl group (R²) and the p-fluorophenyl system (R³) (Figure and Scheme ). Amino derivative 85 was synthesized from cyclohexenecarbaldehyde 62 via reductive amination with methylamine, followed by reduction of the nitro group. For analogues 86 and 87, intermediate 62 was treated with sodium borohydride to reduce the aldehyde to the corresponding alcohol. This transformation occurred without epimerization at the NO₂-substituted carbon. Subsequent treatment with pyridine promoted the epimerization, affording stereoisomer 91, which exhibits the same relative configuration as all final compounds. Next, Mitsunobu reaction between alcohol 91 and phthalimide yielded intermediate 92, which was then converted into primary amine 93. From this intermediate, reduction of the nitro group provided final compound 86, while target acetamide 87 was prepared by acetylation followed by reduction. Regarding the oxygen derivatives (Scheme ), amino alcohol 88 was obtained by reduction of the nitro group in intermediate 91. N-Boc protection of 88, O-methylation and final deprotection afforded methoxy derivative 89.

4. Synthesis of Compounds 85–89 .

^a Reagents and conditions: (a) i. methylamine, MeOH, rt, 3 h; ii. NaBH₄, rt, 3 h, 58%; (b) Zn, AcOH/MeOH, 2 h, 51–91%; (c) i. NaBH₄, MeOH, rt, 2 h; ii. pyridine, EtOAc, rt, 24 h, 75%; (d) phtalimide, PPh₃, DEAD, THF, rt, 4 h, 64%; (e) N₂H₄·H₂O, EtOH, reflux, 2 h, 72%; (f) Ac₂O, DCM:py 3:1, 0 °C, 1 h, on, rt, 85%; (b) Boc₂O, Et₃N, DCM, rt, 4 h, 64%; (c) i. CsOH·H₂O, DMF, rt, 30 min; ii. MeI, rt, 4 h, 50%; (d) HCl 4 M in dioxane, rt, on, 63%.

The new compounds 85–89 were assessed for inhibition of SA-β-gal activity and viability in both proliferative and senescent A549 cells, revealing that only analogue 85, bearing a methylamino group, retained the senolytic effect (Table and Figure C). According to these results, compounds 25 and 85, endowed with low SA-β-gal activity (20% and 49% at 10 μM, respectively, Table ) and senolytic activity (ca. 1.3-fold higher viability in proliferative vs senescent A549 cells, Figure C), were assessed for cell permeability. PAMPA revealed limited permeability values (P) of 8.4·10^–6 and 3.4·10^–6 cm/s, respectively. Hence, we synthesized new N-methyl derivatives 97 and 98 that have higher predicted lipophilicity (clogP) than parent analogues 25 and 85 (Table ). For the synthesis of these new compounds, both amino groups present in compounds 25 and 85 were protected with the Boc group, followed by methylation with iodomethane, and final deprotection (Scheme ).

2. Senotherapeutic Activity of Compounds 85–89, 97, 98 in the FDG SA-β-gal Assay and Permeability Values.

^aData are the mean ± SEM of at least three independent experiments performed in triplicate.

^bPAMPA acceptance criteria: P ≤ 10^–7 cm/s for low permeability and P ≥ 10^–5 cm/s for high permeability.

^cValue calculated with the ACD/Laboratories Percepta software (version 6.0).

5. Synthesis of Compounds 97 and 98 .

^a Reagents and conditions: (a) Boc₂O, Et₃N, DCM, rt, 4 h, 66–84%; (b) i. NaH, DMF, 0 °C, 1 h; ii. MeI, rt, on, 78%; (c) HCl 4 M in dioxane, rt, on, 81–90%.

Compound 98 did not exhibit an improved permeability but retained SA-β-gal activity and moreover it showed the highest selectivity for clearing senescent vs proliferative A549 cells (1.5 fold, Figure C). Taken together, the data obtained in the in vitro senescence models and the permeability assay (Table and Figure C) led to the selection of compounds 25 and 98. The IC₅₀ values, determined from the concentration–response curves (0.4 ± 0.1 μM for 25 and 1.4 ± 0.5 μM for 98, Figure S1), confirmed a potent SA-β-gal inhibition.

Next, we evaluated additional ADME properties. Metabolic stability in mouse (MLM) and human (HLM) liver microsomes was determined and high half-life times (t _1/2) indicated good first-pass metabolism for both compounds (Table ). Also, in human and mouse serum t _1/2 higher than 48 h were obtained. Binding to HSA protein revealed values around 70% indicating an appropriate capacity of the compounds to be distributed from plasma to cells. In addition, none of the compounds showed relevant inhibition of the hERG channel (IC₅₀ >100 μM), suggesting a low potential for cardiotoxicity.

3. ADME Properties and In Vivo Pharmacokinetic Parameters Obtained for Compounds 25 and 98 .

^aData are the mean ± SEM of at least two experiments performed in duplicate.

^bParameters obtained from lineal regression: ln­([compound]) = ln­(C ₀) – k _e·t. V _D: distribution volume; k _e: elimination rate constant; AUC: area under the curve; CL: clearance.

In Vivo Efficacy of the New Senolytic Compounds 25 and 98 in a Mouse Pulmonary Fibrosis Model

In vivo pharmacokinetics was addressed for selected compounds 25 and 98. Following a single intraperitoneal (i.p.) administration of a 40 mg/kg dose to healthy male and female mice, plasma concentrations were quantifiable up to 6 h. The obtained t _1/2 values of ca. 2 h for 25 and longer than 3 h for analogue 98 (Table ) allowed for the assessment of efficacy in vivo. The effect of compounds 25 (UCM-17017) and 98 (UCM-7221) was tested in a bleomycin-triggered mouse model of pulmonary fibrosis. After 2 weeks of intratracheal administration of bleomycin, animals (n = 8) were treated intraperitoneally with each tested compound (40 mg/kg) or vehicle for 7 days. The reference senolytic ABT-263 was administered orally (50 mg/kg) to the control group. Lungs were removed at the end of the experiment and were processed for fibrotic and senescence markers. The lung damage caused by bleomycin is linked to weight loss in mice, which was not affected by either compound 25, 98, or ABT-263 (Figure A). Masson trichrome staining in the lungs confirmed that bleomycin treatment causes the accumulation of collagen, leading to fibrotic tissue formation (Figure B, upper panels). Treatment with compound 25 significantly reduced collagen deposition in the lung to levels similar to those seen with the reference senolytic ABT-263. However, no effect was observed for compound 98. SA-β-gal activity was also quantified via histochemical staining of the lungs and a moderate reduction in the senescence marker was observed for compound 98, whereas 25 and ABT-263 showed a nonsignificant trend (Figure B, bottom panels). This observation is consistent with previous reports highlighting the cell-type specificity of ABT-263 senolytic activity. , Likewise, compound 98 may preferentially target a subset of senescent cells that do not play a central role in driving fibrosis in this model. Additionally, we measured transcriptional levels of markers for senescence (Cdkn1a), fibrosis (Col1a1) and inflammation (Ccl2), as shown in Figure C. Compound 25 was able to reduce the expression levels of Ccl2, a key marker of inflammation and extracellular matrix remodeling, which plays a pivotal role in senescence and fibrosis. , Importantly, hematoxylin-eosin (H&E) staining and the absence of cleaved caspase-3 expression in liver and kidney tissues collected from treated mice indicated a lack of treatment-induced structural damage, apoptosis or toxicity in both key metabolic organs (Figure S2). These preliminary data support the safety profile of compound 25 at a dose of 40 mg/kg in vivo.

4.

In vivo characterization of compounds 25 and 98 in a pulmonary fibrosis mouse model. (A) Body weight measurement of mice throughout the experiment (Ctrol: control group; ABT: ABT-263 treated group; Comp 25: compound 25 treated group; Comp 98: compound 98 treated group). (B) Immunohistochemical images of lungs stained for collagen deposition by Masson trichrome (top left) and SA-β-gal (bottom left) staining, and corresponding quantifications (right). (C) mRNA expression (relative to Gapdh) of the senescence marker Cdkn1a (left), the fibrosis marker Col1a1 (middle), and the inflammation marker Ccl2 (right). Statistical significance was assessed by the Mann–Whitney test: *p < 0.05, **p < 0.01, ***p < 0.001.

Taken together, compound 25 shows cellular senolytic activity, and its in vivo effect in the bleomycin-induced model primarily involves the modulation of the pro-inflammatory and pro-fibrotic environment. These results show the efficacy of the new senolytic agent in targeting pulmonary fibrosis.

Conclusion

In the screening of our in-house chemical library in relevant cellular senescence phenotypes, we identified tetrasubstituted cyclohexene scaffold as a new bioactive chemotype. Following the synthesis and evaluation of related analogues, we have discovered compound 25 (UCM-17017) that decreases SA-β-gal activity in senescent human fibroblasts, reduces the viability of senescent human lung adenocarcinoma (A549) cells over proliferative cells, and exhibits a moderate in vitro ADME profile, which translated into favorable pharmacokinetics in vivo. In a mouse model of pulmonary fibrosis, the new senolytic compound UCM-17017 induces a significant reduction in collagen deposition and in the expression levels of the inflammation marker Ccl2. Our results reinforce the targeting of cellular senescence as a promising strategy to provide new therapies against pulmonary fibrosis, one of the most common diseases directly related to the senescence process and with unmet medical needs.

Experimental Section

Synthesis

The starting materials, reagents, and solvents were purchased as high-grade commercial products from Sigma-Aldrich (Merck), Fisher, Acros, ABCR, Fluorochem, or Scharlab. Dichloromethane (DCM), tetrahydrofuran (THF) and diethyl ether were dried using a Pure Solv Micro 100 Liter solvent purification system. Ethanol-free chloroform was obtained by washing with water and subsequent distillation over P₂O₅.

Analytical thin-layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel 60 F-254), with detection by UV light (λ = 254 nm), 5% ninhydrin solution in ethanol, or 10% phosphomolybdic acid solution in ethanol. Unless otherwise stated, products were purified in a Biotage Selekt system using silica gel cartridges (Biotage Sfär, size particle 60 μM).

All compounds were obtained as oils, except those whose melting points (mp) are indicated, which were solids. Mp were determined on a Stuart Scientific electrothermal apparatus. Infrared (IR) spectra were measured on a Bruker Tensor 27 instrument equipped with a Specac attenuated total reflection (ATR) accessory of 5200–650 cm^–1 transmission range; frequencies (ν) are expressed in cm^–1. ¹H- and ¹³C NMR spectra were recorded on a Bruker Avance III 700 MHz (¹H, 700 MHz; ¹³C, 175 MHz), Bruker Avance 500 MHz (¹H, 500 MHz; ¹³C, 125 MHz) or Bruker DPX 300 MHz (¹H, 300 MHz; ¹³C, 75 MHz) instrument at rt at the Universidad Complutense de Madrid (UCM) NMR facility. Bruker DPX 300 MHz spectrometer was used unless otherwise stated. Chemical shifts (δ) are expressed in parts per million relative to the residual solvent peak for ¹H and ¹³C nuclei (CDCl₃: δ_H = 7.26, δ_C = 77.16; MeOH-d4: δ_H = 3.31, δ_C = 49.00) and to internal (trifluoromethyl)­benzene for ¹⁹F nucleus; coupling constants (J) are in hertz (Hz). The following abbreviations are used to describe peak patterns when appropriate: s (singlet), d (doublet), t (triplet), q (quadruplet), quint (quintuplet), m (multiplet), and br (broad). 2D NMR experimentshomonuclear correlation spectroscopy (H,H–COSY), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC)of representative compounds were acquired to assign protons and carbons of new structures. The following abbreviations have been used for the peak assignment: cpr (cyclopropane), ind (indole), py (pyridine), thz (thiazole), morp (morpholine), and ox (oxazole). The relative configuration of the compounds was confirmed by 1D ¹H NMR NOE experiments, in which the signal of interest was irradiated with a selective pulse and NOE interactions were observed. Numbered chemical structures for NMR assignation of compounds 1, 25, 34, 35, 62, 77, 98, 100, and 102 described in this section are shown in Figure S3. High-resolution mass spectrometry (HRMS) was carried out on Bruker Impact II QTOF mass spectrometer in electrospray ionization (ESI) mode at UCM’s mass spectrometry facilities.

For all final compounds, a purity of at least 95% was determined by HPLC-MS using an Agilent 1200LC-MSD VL instrument. LC separation was achieved with a Zorbax SB-C3 column (5 μm, 2.1 mm × 50 mm) or an Eclipse XDB-C18 column (5 μm, 4.6 mm × 150 mm), together with a guard column (5 μm, 4.6 mm × 12.5 mm). The mobile phase consisted of water and acetonitrile (ACN) with 0.1% formic acid as solvent modifier, and the gradients are indicated in Table S2. MS analysis was performed using an electrospray irradiation source. The capillary voltage was set to 3.0 kV and the fragmentor voltage to 72 eV. The drying gas temperature was 350 °C, the drying gas flow was 10 L/min, and the nebulizer pressure was 20 psi. Spectra were acquired in positive or negative ionization mode from 80 to 800 m/z and in UV-mode at four different wavelengths (210, 230, 254, and 280 nm).

Optical rotation [α] was measured on an Anton Paar MCP 100 modular circular polarimeter using a sodium lamp (λ = 589 nm) and a 0.1 dm path length; concentrations (c) are given in g/100 mL. The enantiomeric ratio (er) of final compounds 25 and 98 was determined by chiral HPLC using Chiralpak IA and IC columns. HPLC traces were compared with those obtained from the corresponding racemic samples. The enantiomers of 25 and 98 were prepared following the same synthetic route, employing the S-enantiomer of the Jørgensen–Hayashi catalyst in the initial asymmetric cascade forming the cyclohexene scaffold.

Final compounds 1–33, 85–89, 97 and 98 were characterized (α, R_f, IR, NMR, HPLC-MS) and subsequently transformed into the corresponding hydrochloride salts. Thus, a commercial solution of 2 M HCl (g) in diethyl ether (3 mL/mmol) was added to a solution of the compound in anhydrous DCM or methanol (MeOH) (6 mL/mmol). The resulting salt was isolated by filtration or evaporation of the solvent, washed with anhydrous diethyl ether, and dried under vacuum. A purity of at least for 95% for the salts was determined by HPLC-MS and elemental chemical analysis (C, H, N, S) using a LECO CHNS-932 instrument at the UCM Elemental Analysis facility.

IUPAC rules have been followed to name all organic compounds, except for 1-methoxy-4-[(E)-2-nitroethenyl]­benzene and pyridine-2-carboxylic, pyridine-3-carboxylic, pyridine-4-carboxylic and pentanoic acids, whose common names trans-p-methoxy-β-nitrostyrene and picolinic, nicotinic, isonicotinic and valeric acids, respectively, have been employed for simplicity.

No unexpected or unusually high safety hazards were encountered during the course of the experimental work reported.

General Procedure A: Three-Component Cascade Reaction

To a solution of the corresponding nitroalkene (1.00 equiv) and (R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine (0.20 equiv) in anhydrous toluene (0.8 mL/mmol) at 0 °C, propionaldehyde (1.20 equiv) and acrolein (1.05 equiv) were added successively. After stirring at 0 °C for 1 h, the reaction was warmed up to rt and stirred overnight. Then, the solvent was removed under reduced pressure and the residue was purified by flash chromatography to afford the corresponding cyclohexenecarbaldehyde 34, 55–69.

(1R,2R,6R)-4′-Methoxy-6-methyl-2-nitro-1,2,3,6-tetrahydro­[1,1′-biphenyl]-4-carbaldehyde, 34

Following general procedure A using trans-p-methoxy-β-nitrostyrene (550 mg, 3.07 mmol), compound 34 was obtained as a yellow solid (423 mg, 50%). Chromatography: hexane to hexane/EtOAc 7:3. Mp: 126–128 °C. R_f: 0.33 (hexane/EtOAc 7:3). [α]₂₀ ^D = −12.9 (c = 1.10, CHCl₃). IR (ATR): ν 1681 (CO), 1546 (NO₂), 1251 (COC). ¹H NMR (CDCl₃, 700 MHz): δ 1.22 (d, J = 7.2, 3H, CH₃), 2.80 (dt, J = 5.4, 1.7, 2H, 2H₃), 3.12 (dd, J = 6.9, 3.8, 1H, H₁), 3.16–3.21 (m, 1H, H₆), 3.79 (s, 3H, OCH₃), 4.90 (td, J = 5.6, 3.8, 1H, H₂), 6.84 (d, J = 8.7, 2H, H_3′, H_5′), 6.91 (dt, J = 3.2, 1.7, 1H, H₅), 7.00 (d, J = 8.7, 2H, H_2′, H_6′), 9.57 (s, 1H, CHO). ¹³C NMR (CDCl₃, 175 MHz): δ 19.9 (CH₃), 24.6 (C₃), 34.4 (C₆), 48.9 (C₁), 55.4 (OCH₃), 83.9 (C₂), 114.5 (C_3′, C_5′), 128.8 (C_2′, C_6′), 129.5 (C_1′), 136.0 (C₄), 154.2 (C₅), 159.5 (C_4′), 192.6 (CHO). 1D ¹H NMR NOE: irradiation of the signal at δ 3.16–3.21 ppm (m, H₆) yielded NOE on 7.00 (d, H_2′, H₆); and irradiation of the signal at δ 4.90 ppm (td, H₂) yielded NOE on 3.12 (dd, H₁). HPLC (method A, t_R, min): 12.9. MS (ESI, m/z, %): 274.1 ([M-H]⁻, 100).

(1R,2R,6R)-4′-Fluoro-6-methyl-2-nitro-1,2,3,6-tetrahydro­[1,1′-biphenyl]-4-carbaldehyde, 62

Following general procedure A using 1-fluoro-4-[(E)-2-nitroethenyl]­benzene (400 mg, 2.40 mmol), compound 62 was obtained as a yellow oil (198 mg, 31%). Chromatography: hexane to hexane/EtOAc 6:4. R_f: 0.43 (hexane/EtOAc 7:3). [α]₂₀ ^D = −73.0 (c = 1.0, CHCl₃). IR (ATR): ν 1682 (CO), 1510 (NO₂). ¹H-RMN (CDCl₃): δ 1.22 (d, J = 6.9, 3H, CH₃), 2.81 (dt, J = 6.4, 1.7, 2H, 2H₃), 3.13–3.22 (m, 2H, H₁, H₆), 4.91 (td, J = 5.5, 3.4, 1H, H₂), 6.89–6.91 (m, 1H, H₅), 7.01–7.07 (m, 4H, H_2′, H_3′, H_5′, H_6’), 9.57 (s, 1H, CHO). ¹³C-RMN (CDCl₃): δ 19.8 (CH₃), 24.6 (C₃), 34.2 (C₆), 48.9 (C₁), 83.7 (C₂), 116.1 (d, J = 21.4, C_3′, C_5′), 129.4 (d, J = 8.1, C_2′, C_6′), 133.4 (d, J = 3.4, C_1′), 135.9 (C₄), 153.7 (C₅), 162.6 (d, J = 247.3, C_4′), 192.5 (CHO). HPLC (method B, t_R, min): 10.50.

General Procedure B: Four-Component Cascade Reaction

To a solution of (R)-2-{diphenyl­[(trimethylsilyl)­oxy]­methyl}­pyrrolidine (0.20 equiv), the corresponding nitroalkene (1.00 equiv) and nucleophile (1.20 equiv), and benzoic acid (0.25 equiv) in ethanol-free chloroform (1 mL/mmol), a 1 M solution of acrolein in ethanol-free chloroform (3.00 equiv) was added via a syringe pump (0.9 mL/h). After addition was complete, the reaction mixture was stirred at rt overnight. Then, the solvent was removed under reduced pressure and the residue was purified by flash chromatography to afford the corresponding cyclohexenecarbaldehyde 45–49.

General Procedure C: One-Pot Reductive Amination

To a solution of the corresponding aldehyde (1.00–1.50 equiv) in anhydrous methanol (5 mL/mmol) and DCM (2 mL/mmol; only in those cases where the aldehyde is not soluble in methanol) under nitrogen atmosphere, the appropriate amine (1.00–2.00 equiv) was added and the reaction mixture was stirred at rt for 2–4 h to form the corresponding imine (confirmed by ¹H NMR analysis of an aliquot). Then, NaBH₄ (2.00 equiv) was added at 0 °C and the mixture was allowed to react at rt for 3 h. The reaction was quenched with a sat. NaHCO₃ solution and the solvent was evaporated under reduced pressure. The residue was suspended in water and extracted with EtOAc (×2). The organic layers were washed with brine, dried over Na₂SO₄, filtered, and evaporated under reduced pressure to afford the corresponding amine 35, 50–54, 70–84, 90, which was purified by flash chromatography or used in the next step without further purification.

1-Cyclopropyl-N-{[(1R,2S,6R)-4′-methoxy-6-methyl-2-nitro-1,2,3,6-tetrahydro­[1,1′-biphenyl]-4-yl]­methyl}­methanamine, 35

Following general procedure C using 34 (450 mg, 1.64 mmol) and (cyclopropylmethyl)­amine (0.28 mL, 3.28 mmol), compound 35 was obtained as a yellow oil (257 mg, 57%). Chromatography: DCM to DCM/MeOH/NH₃ 9:1:0.1. R_f: 0.48 (DCM/MeOH/NH₃ 9:1:0.1). [α]₂₀ ^D = −4.9 (c = 0.62, CHCl₃). IR (ATR): ν 3311 (NH), 1550 (NO₂), 1251 (COC). ¹H NMR (CDCl₃, 700 MHz): δ 0.12–0.14 (m, 2H, CH_2cpr), 0.49–0.52 (m, 2H, CH_2cpr), 0.90 (d, J = 7.0, 3H, CH₃), 0.94–1.00 (m, 1H, CH_cpr), 1.73 (br s, 1H, NH), 2.44–2.48 (m, 3H, H₆, NHCH ₂CH), 2.72–2.84 (m, 2H, 2H₃), 2.82 (t, J = 11.1, 1H, H₁), 3.24 (AB system, J = 13.8, 2H, NHCH ₂), 3.78 (s, 3H, OCH₃), 4.96 (td, J = 11.1, 5.5, 1H, H₂), 5.56 (s, 1H, H₅), 6.83 (d, J = 8.2, 2H, H_3′, H_5′), 7.10 (d, J = 8.3, 2H, H_2′, H_6′). ¹³C NMR (CDCl₃, 175 MHz): δ 3.6 (2CH_2cpr), 11.3 (CH_cpr), 19.7 (CH₃), 33.4 (C₃), 37.5 (C₆), 51.8 (C₁), 54.6 (NHCH₂CH), 54.8 (NHCH₂), 55.3 (OCH₃), 88.8 (C₂), 114.2 (C_3′, C_5′), 128.4 (C₅), 129.1 (C_2′, C_6′), 130.6 (C_1′), 131.8 (C₄), 159.0 (C_4′). 1D ¹H NMR NOE: irradiation of the signal at δ 0.90 ppm (d, CH₃) yielded NOE on 2.82 (t, H₁); irradiation of the signal at δ 2.44–2.48 ppm (m, H₆) yielded NOE on 7.10 (d, H_2′, H_6′); and irradiation of the signal at δ 4.96 ppm (td, H₂) yielded NOE on 7.10 (d, H_2′, H_6′). HPLC (method A, t_R, min): 15.25. MS (ESI, m/z, %): 331.1 ([M + H]⁺, 100).

1-Cyclopropyl-N-{[(1R,2S,6R)-4′-fluoro-6-methyl-2-nitro-1,2,3,6-tetrahydro­[1,1′-biphenyl]-4-yl} methanamine, 77

Following general procedure C using 62 (188 mg, 0.71 mmol) and (cyclopropylmethyl)­amine (0.12 mL, 1.42 mmol), compound 77 was obtained as a yellow oil (102 mg, 45%). Chromatography: DCM to DCM/EtOH/NH₃ 9:1:0.1. R_f: 0.69 (DCM/MeOH/NH₃ 9:1:0.1). [α]₂₀ ^D = −3.0 (c = 1.0, CHCl₃). IR (ATR): ν 3311 (NH₂), 1513 (NH), 1549 (NO₂). ¹H-RMN (CDCl₃): δ 0.01–0.05 (m, 2H, CH_2cpr), 0.37–0.43 (m, 2H, CH_2cpr), 0.79 (d, J = 7.0, 3H, CH₃), 0.84–0.92 (m, 1H, CH_cpr), 1.75 (br s, 1H, NH), 2.40–2.54 (m, 3H, H₆, NHCH ₂CH), 2.78 (dd, J = 8.6, 1.8, 2H, 2H₃), 2.77 (dd, J = 11.5, 10.5, 1H, H₁), 3.15 (s, 2H, NHCH ₂), 4.86 (ddd, J = 11.6, 9.1, 7.2, 1H, H₂), 5.56 (d, J = 1.1, 1H, H₅), 6.87–6.92 (m, 2H, H_3′, H_5′), 7.05 (dd, J = 8.7, 5.3, 2H, H_2′, H_6′). ¹³C-RMN (CDCl₃): δ 3.56 (CH_2cpr), 3.57 (CH_2cpr), 11.3 (CH_cpr), 19.6 (CH₃), 33.3 (C₃), 37.5 (C₆), 51.8 (C₁), 54.5 (NHCH₂CH), 54.7 (NHCH₂), 88.6 (C₂), 115.8 (d, J = 21.4, C_3′, C_5′), 128.2 (C₅), 129.6 (C_2′, C_6′), 131.9 (C₄)_, 134.4 (C_1′), 162.3 (d, J = 246.0, C_4′). HPLC (method A, t_R, min): 14.90. MS (ESI, m/z, %): 318.9 ([M + H]⁺, 100).

General Procedure D: Nitro Group Reduction with Zn/Acetic Acid

To a solution of the corresponding nitro derivative (1.00 equiv) in a 1:1 mixture of glacial acetic acid and anhydrous methanol (4 mL/mmol), Zn powder (10.0 equiv) was added and the reaction was stirred until complete conversion of starting material (1–3 h). Then, the mixture was filtered and washed with methanol, and the filtrate was evaporated. The residue was suspended in a sat. NaHCO₃ solution and extracted with DCM (×3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography to afford the corresponding final compound 1, 13–17, 18–32, 85–87, or intermediate 37, 88.

(1R,2S,6R)-4-{[(Cyclopropylmethyl)­amino]­methyl}-4′-methoxy-6-methyl-1,2,3,6-tetrahydro [1,1′-biphenyl]-2-amine, 1

Following general procedure D using 35 (216 mg, 0.65 mmol), compound 1 was obtained as a yellow oil (176 mg, 90%, er >95:5). Chromatography: DCM to DCM/MeOH/NH₃ 8:2:0.1. R_f: 0.04 (DCM/MeOH/NH₃ 9:1:0.1). [α]₂₀ ^D = −12.2 (c = 0.99, CHCl₃). ¹H NMR signal in the diastereomer from chiral derivatization reaction: δ 6.01 ppm. IR (ATR): ν 3560 (NH₂), 1513 (C–N), 1250 (COC). ¹H NMR (CDCl₃, 700 MHz): δ 0.11–0.13 (m, 2H, CH_2cpr), 0.48–0.50 (m, 2H, CH_2cpr), 0.81 (d, J = 7.0, 3H, CH₃), 0.96–1.01 (m, 1H, CH_cpr), 1.93–1.97 (m, 1H, H₁), 2.00 (t, J = 10.4, 1H, H₃), 2.35–2.38 (m, 2H, H₃, H₆), 2.46 (dd, J = 7.3, 2.5, 2H, NHCH ₂CH), 3.16 (td, J = 10.5, 5.2, 1H, H₂), 3.21 (AB system, J = 13.8, 2H, NHCH ₂), 3.80 (s, 3H, OCH₃), 5.47 (s, 1H, H₅), 6.87 (d, J = 8.3, 2H, H_3′, H_5′), 7.11 (d, J = 8.3, 2H, H_2′, H_6′).¹³C NMR (CDCl₃, 175 MHz): δ 3.5 (CH_2cpr), 3.6 (CH_2cpr), 11.4 (CH_cpr), 20.2 (CH₃), 37.0 (C₃), 38.1 (C₆), 51.9 (C₂), 54.5 (NHCH₂CH), 55.3 (NHCH₂), 55.4 (OCH₃), 57.4 (C₁), 114.1 (C_3′, C_5′), 128.5 (C₅), 129.4 (C_2′, C_6′), 133.7 (C_1′), 134.7 (C₄), 158.4 (C_4′). 1D ¹H NMR NOE: irradiation of the signal at δ 3.16 ppm (td, H₂) yielded NOE on 7.11 (d, H_2′, H_6′). HPLC for HCl salt (method C, t_R, min): 4.00 MS (ESI, m/z, %): 301.1 ([M + H]⁺, 100). Elemental analysis calculated for C₁₉H₂₈N₂O·2HCl·2H₂O: %C 55.74, %H 8.37, %N 6.84; experimental: %C 56.13, %H 7.99, %N 6.49.

(1R,2S,6R)-4-{[(Cyclopropylmethyl)­amino]­methyl}-4′-fluoro-6-methyl-1,2,3,6-tetrahydro­[1,1′-biphenyl]-2-amine, 25 (UCM-17017)

Following general procedure D using 77 (86 mg, 0.27 mmol), compound 25 was obtained as a yellow oil (55 mg, 71%, er >95:5). Chromatography: DCM to DCM/EtOH/NH₃ 8:2:0.1. R_f: 0.37 (DCM/MeOH/NH₃ 9:1:0.1). [α]₂₀ ^D = −11.0 (c = 1.0, CHCl₃). IR (ATR): ν 3300 (NH₂). ¹H NMR (CDCl₃, 500 MHz): δ 0.11–0.12 (m, 2H, CH_2cpr), 0.46–0.48 (m, 2H, CH_2cpr), 0.77 (d, J = 7.0, 3H, CH₃), 0.94–0.98 (m, 1H, CH_cpr), 1.84–1.98 (m, 4H, H₃, NH, NH₂), 2.04 (t, J = 10.4, 1H, H₁), 2.31–2.40 (m, 2H, H₃, H₆), 2.45 (d, J = 6.8, 2H, NHCH ₂CH), 3.15 (td, J = 10.5, 5.2, 1H, H₂), 3.20 (d, J = 5.1, 2H, NHCH ₂), 5.45 (s, 1H, H₅), 6.99 (t, J = 8.4, 2H, H_3′, H_5′), 7.13 (dd, J = 8.4, 5.4, 2H, H_2′, H_6′). ¹³C NMR (CDCl₃, 125 MHz): δ 3.5, 3.6 (2CH_2cpr), 11.1 (CH_cpr), 20.0 (CH₃), 36.8 (C₃), 38.1 (C₆), 51.8 (C₂), 54.3 (NHCH₂CH), 55.0 (NHCH₂), 57.3 (C₁), 115.5 (d, J = 21.0, C_3′, C_5′), 128.6 (C₅), 129.8 (d, J = 7.6, C_2′, C_6′), 133.4 (C₄), 138.3 (d, J = 3.3, C_1′), 161.7 (d, J = 244.4, C_4′). HPLC for HCl salt (method C, t_R, min): 4.77. MS (ESI, m/z, %): 289.2 ([M + H]⁺, 100). HRMS (ESI, m/z): calculated for C₁₈H₂₆FN₂ [M + H]⁺: 289.2075, found: 289.2069. Elemental analysis calculated for C₁₈H₂₅FN₂·2HCl·3H₂O: %C 52.05, %H 8.01, %N 6.55; experimental: %C 52.39, %H 7.83, %N: 6.55.

General Procedure E: Amidation Reaction

A solution of the appropriate carboxylic acid (1.00 equiv), HOBt (1.10 equiv), EDC (1.10 equiv) and DIPEA (1.10 equiv) in anhydrous DCM (5 mL/mmol) under nitrogen atmosphere was stirred at rt until complete consumption of starting material (40 min–3 h). Then, a solution of the corresponding amine (1.00 equiv) in anhydrous DCM (5 mL/mmol) was added at 0 °C and the reaction was stirred at this temperature for 3 h. Next, the mixture was diluted with EtOAc, and successively washed with water, a 1 M K₂CO₃ solution, and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to afford the corresponding N-Fmoc amide derivative, intermediate 44, or final compound 2, 3.

General Procedure F: N-Fmoc Deprotection

To a solution of the corresponding N-Fmoc-protected amine (1.00 equiv) in anhydrous DCM (10 mL/mmol), piperidine (10.0 equiv) was added and the reaction was stirred at rt until complete consumption of starting material (8–16 h). The reaction was diluted with DCM and washed with water (×2). The organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to afford the corresponding final compound 4–7.

General Procedure G: N-Boc Protection

To a solution of the corresponding aminoderivative (1.00 equiv) and triethylamine in anhydrous DCM (7 mL/mmol) at 0 °C, a solution of di-tert-butyl dicarbonate (2 or 4 equiv) in DCM (3 mL/mmol) was added dropwise and the reaction mixture was stirred at 0 °C for 30 min and at rt until complete conversion of starting material (1–4 h). Then, the solvent was removed under reduced pressure and the residue was purified by flash chromatography to afford the corresponding N-Boc intermediate 38, 95, 99, 100.

tert-Butyl ({(1R,2S,6R)-2-[(tert-Butoxycarbonyl)­amino]-4′-fluoro-6-methyl-1,2,3,6-tetrahydro­[1,1′-biphenyl]-4-yl}­methyl)­methylcarbamate, 100

Following general procedure G using 85 (130 mg, 0.52 mmol), triethylamine (0.2 mL, 1.36 mmol) and di-tert-butyl dicarbonate (457 mg, 2.08 mmol), compound 100 was obtained as a colorless oil (196 mg, 84%). Chromatography: hexane to hexane/EtOAc 7:3. R_f: 0.70 (hexane/EtOAc 7:3). [α]₂₀ ^D = −19.0 (c = 1.0, CHCl₃). IR (ATR): ν 3362 (NH), 1696 (CO), 1511 (C–N). ¹H NMR (CDCl₃, mixture of rotamers): δ 0.82 (d, J = 7.0, 3H, CH₃), 1.24 (s, 9H, 3CH₃), 1.46 (s, 9H, 3CH₃), 1.85–1.97 (m, 1H, H₃), 2.21 (t, J = 8.0, 1H, H₁), 2.44 (br s, 2H, H₃, H₆), 2.81 (br s, 3H, NCH₃), 3.75 and 3.96 (br s, 2H, NCH₂), 4.13 (br s, 1H, H₂), 5.37 (s, 1H, H₅), 6.98 (t, J = 8.7, 2H, H_3′, H_5′), 7.14 (dd, J = 8.5, 5.5, 2H, H_2′, H_6′). ¹³C NMR (CDCl₃): δ 20.2 (CH₃), 28.3 (3CH₃), 28.6 (3CH₃), 33.7 (NCH₃), 34.7 (br, C₃), 38.6 (C₆), 51.1 (br, C₂), 53.3 and 54.3 (br, NCH₂), 54.6 (br, C₁), 79.2 (C­(CH₃)₃), 79.7 (C­(CH₃)₃), 115.2 (d, J = 21.4, C_3′, C_5′), 129.0 (br, C₅), 130.1 (d, J = 7.8, C_2′, C_6′), 131.5 (C₄), 137.2 (br, C_1′), 155.2 (CO), 156.2 (CO), 161.9 (d, J = 244.1, C_4′). HPLC (method B, t_R, min): 12.36. MS (ESI, m/z, %): 349.2 ([M-Boc]⁺, 100).

General Procedure H: N-Methylation

To a solution of the corresponding amine (1.00 equiv) in anhydrous DMF (7 mL/mmol) at 0 °C, NaH (4.00 equiv) was added and the reaction was stirred for 1 h. Then, iodomethane (10.0 equiv) was added and the mixture was stirred at rt overnight. The reaction was quenched with a sat. NaHCO₃ solution and extracted with EtOAc (×2). The organic layers were washed with brine, dried over Na₂SO₄, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography to afford the corresponding N-methyl derivative 101, 102.

tert-Butyl [(1R,2S,6R)-4-{[(tert-Butoxycarbonyl)­(cyclopropylmethyl)­amino]­methyl}-4′-fluoro-6-{[(pyridin-2-yl)­methoxy]­methyl}-1,2,3,6-tetrahydro­[1,1′-biphenyl]-2-yl]­methylcarbamate, 102

Following general procedure H, using 100 (196 mg, 0.44 mmol), compound 102 was obtained as a colorless oil (157 mg, 78%). Chromatography: hexane to hexane/EtOAc 7:3. R_f: 0.30 (hexane/EtOAc 7:3). [α]₂₀ ^D = −19.0 (c = 1.0, CHCl₃). IR (ATR): ν 1691 (CO), 1511 (C–N). ¹H NMR (CDCl₃, mixture of rotamers): δ 0.81 (d, J = 6.9, 3H, CH₃), 1.26 (s, 9H, 3CH₃), 1.46 (s, 9H, 3CH₃), 1.98–2.09 (m, 1H, H₃), 2.10–2.23 (m, 1H, H₃), 2.25–2.40 (m, 2H, H₁, H₆), 2.43 and 2.51 (s, 3H, CHNCH ₃), 2.82 (br s, 3H, CH₂NCH ₃), 3.61–4.00 (br m, 2H, NCH₂), 4.49–4.58 and 4.74–4.84 (m, 1H, H₂), 5.34 (s, 1H, H₅), 6.93–6.97 (m, 2H, H_3′, H_5′), 7.06–7.11 and 7.14–7.17 (m, 2H, H_2′, H_6′). ¹³C NMR (CDCl₃): δ 20.1 (CH₃), 27.3 and 27.9 (CHNCH₃), 28.4 (3CH₃), 28.6 (3CH₃), 29.8 and 30.1 (C₃), 33.7 and 33.8 (CH₂NCH₃), 39.26 and 39.33 (C₆), 51.7 (C₁), 53.5 and 54.4 (NCH₂), 53.7 and 55.0 (C₂), 79.6 (C­(CH₃)₃), 79.7 (C­(CH₃)₃), 114.9 and 115.2 (d, J = 21.2, C_3′, C_5′), 129.0 (br, C₅), 129.6 and 130.0 (C_2′, C_6′), 131.8 (br, C₄), 137.1 (br, C_1′), 155.4 and 155.9 (CO), 156.2 (br, CO), 161.7 (d, J = 243.6, C_4′). HPLC (method B, t_R, min): 16.90. MS (ESI, m/z, %): 363.3 ([M-Boc]⁺, 100).

General Procedure I: N-Boc Deprotection

A solution of the corresponding N-Boc-protected amine (1.00 equiv) in HCl (4 M in dioxane, 15.0 equiv) was stirred at rt until complete consumption of starting material (4–16 h). Solvent was then evaporated under reduced pressure and the corresponding final compound 8–12, 89, 97, 98 was purified using a SiliCycle SCX-2 cartridge and elution with 2 M NH₃ in methanol according to the manufacturer procedure.

(1R,2S,6R)-4′-Fluoro-N,6-dimethyl-4-[(methylamino)­methyl]-1,2,3,6-tetrahydro­[1,1′-biphenyl]-2-amine, 98 (UCM-17221)

Following general procedure I using 102 (160 mg, 0.32 mmol), compound 98 was obtained as a colorless oil (82 mg, 90%, er >95:5). R_f: 0.30 (DCM/MeOH/NH₃ 8:2:0.1). [α]₂₀ ^D = 6.0 (c = 1.00, MeOH). IR (ATR): ν 3328 (NH), 1508 (C–N). ¹H NMR (CDCl₃): δ 0.78 (d, J = 6.9, 3H, CH₃), 1.85–2.00 (m, 1H, H₃), 2.17–2.29 (m, 6H, CHNHCH ₃, H₁, 2NH), 2.30–2.39 (m, 1H, H₆), 2.44 (s, 3H, CH₂NHCH ₃), 2.51 (dd, J = 16.5, 5.0, 1H, H₃), 2.86 (td, J = 10.3, 5.2, 1H, H₂), 3.20 (s, 2H, NHCH ₂), 5.50 (s, 1H, H₅), 7.00 (t, J = 8.7, 2H, H_3′, H_5′), 7.16 (dd, J = 8.7, 5.4, 2H, H_2′, H_6′). ¹³C NMR (CDCl₃): δ 20.0 (CH₃), 33.6 (C₃), 33.9 (CHNHCH₃), 35.5 (CH₂NHCH₃), 38.1 (C₆), 54.7­(C₁), 57.3 (NHCH₂), 59.5 (C₂), 115.7 (d, J = 22.0, C_3′, C_5′), 129.5 (C₅), 129.8 (d, J = 7.7, C_2′, C_6′), 132.4 (C₄), 137.9 (d, J = 3.3, C_1′), 161.9 (d, J = 244.7, C_4′). HPLC for HCl salt (method C, t_R, min): 4.77. MS (ESI, m/z, %): 263.1 ([M + H]⁺, 100). HRMS (ESI, m/z): calculated for C₁₆H₂₄FN₂ [M + H]⁺: 263.1918; found: 263.1913. Elemental analysis calculated for C₁₆H₂₃FN₂·2HCl·2H₂O: %C 51.76, %H 7.87, %N 7.54; experimental: %C 51.92, %H 7.51, %N 7.32.

In Vitro Assays

Cell Lines and Culture

Fibroblast cell line (IMR-90) was obtained from American Type Culture Collection (ATCC; reference number CCL-186) and maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 15% heat-inactivated fetal bovine serum (FBS), 10 U/mL penicillin, and 10 μg/mL streptomycin. A549 cells were cultured in high-glucose DMEM (4,500 mg·L^–1; Sigma-Aldrich), supplemented with 10% FBS (Corning), 1% penicillin/streptomycin (Sigma-Aldrich, P4333), and 1% glutamine (Sigma-Aldrich, G8540), and were routinely tested for mycoplasma contamination. Cells were incubated in a humidified atmosphere at 37 °C in the presence of 5% CO₂.

FDG SA-β-gal Senescence Assay

IMR-90 cells (2 million) were seeded in a P-100 Petri dish with DMEM and left to settle for 24 h. Then, DMEM was replaced by DMEM containing 300 μM H₂O₂. After 2 h, the medium was replaced by fresh DMEM without H₂O₂ and the cells were incubated for 4 days. Next, the cells were split in a 1:2 ratio and incubated for 24 h, followed by a second treatment with DMEM containing 300 μM H₂O₂ for 2 h. Finally, the cells were incubated in fresh DMEM without H₂O₂ for 2–4 days. The senescence phenotype was confirmed both by monitoring the characteristic morphological changes as enlarged and flattered cells with bigger nuclei, and in the FDG SA-β-gal assay.

Senescent IMR-90 cells (6·10³ cells per well) were cultured in DMEM in a 96-well plate overnight for attachment. Then, the medium was replaced by fresh DMEM containing tested compound or the equivalent volume of DMSO as vehicle. After 48 h, the cells were fixed for 5 min in 2% formaldehyde and 0.2% glutaraldehyde buffered with PBS, and then incubated with 95 μL of staining solution (0.2 M citric acid, 0.4 M Na₂HPO₄, 100 mM potassium ferrocyanide, 100 mM potassium ferricyanide, 5 M NaCl, and 0.2 M MgCl₂ in water) and 5 μL of 2 mM FDG (Sigma-Aldrich) for 24 h at 37 °C without CO₂. Then, an equal volume of the supernatant of each well was transferred to a new 96-well plate for fluorescence measurement. Fluorescein fluorescence was registered at 520 nm using a FluoStar Optima instrument (BMG Labtech), with an excitation wavelength of 485 nm. One well containing the reaction mixture without cells was used as blank for subtracting the background fluorescence. For vehicle-treated cells, the higher fluorescence compared to cells not treated with H₂O₂ confirmed the senescence phenotype and was assigned a value of 100% SA-β-gal activity. For compound-treated cells, the SA-β-gal activity was expressed as a fluorescence percentage relative to the vehicle, obtained from at least three experiments performed in triplicate.

For the concentration–response curves, senescent IMR-90 cells were treated with increasing concentrations of the tested compound. IC₅₀ values (n = 3) were determined by nonlinear regression analysis using Prism Software (GraphPad, v.10.4.2).

MTT Viability Assay

IMR-90 cells were seeded in 96-well plates (10·10³ cells per well) in DMEM with 15% FBS for 24 h prior to treatments. The medium was then replaced by fresh medium containing tested compound or the equivalent volume of DMSO as vehicle. After 48 h, the medium was replaced by fresh DMEM with 5 mg/mL of MTT (Sigma-Aldrich), and the cells were incubated for 4 h at 37 °C in the dark. The supernatants were removed, formazan crystals were dissolved in DMSO (100 μL/well), and the absorbance was measured at 570 nm (OD570–630) using an Asys UVM 340 microplate reader (Biochrom Ltd., Cambridge, U.K.). The background absorbance of blank wells containing only medium with compound or vehicle were subtracted from each test well. The results were reported as cell viability percentage for tested compound relative to the vehicle, obtained from at least two experiments performed in triplicate.

Proliferative A549 cells were treated with 20 μM bleomycin (Mylan Pharmaceuticals) for 5 days as a chemotherapeutic agent to induce senescence. Proliferative and senescent A549 cells were treated with the tested compounds for 24, 48, or 72 h and cell viability was determined by MTT assay as above. ABT-263 (Abbvie) was used as a positive control of senolytic compound.

HSA Binding Assay

The compounds were incubated with different concentrations of immobilized HSA, using the TRANSILXL HSA Binding Kit (TMP-0210–2096, Sovicell). An 8-well unit of the TRANSIL assay plate was used for each tested compound; six wells contained increasing concentrations of HSA immobilized on silica beads suspended in PBS at pH 7.4, and two wells contained buffer only to account for nonspecific binding. The TRANSIL assay plate was thawed for 3 h at rt and centrifuged at 750g for 5 s. Then, 15 μL of an 80 μM stock solution of the compound in PBS (for a final concentration of 5 μM) was added to each well, and the plate was incubated on a shaker at 1000 rpm and rt for 12 min. Then the plate was centrifuged at 750g for 10 min, and 50 μL of the supernatants were transferred for analytical quantification by HPLC-MS using selected ion monitoring (SIM) as acquisition method. The binding percentage was calculated from the remaining free compound concentration in the supernatant of each well, using the spreadsheet and algorithms supplied with the kit.

Stability in Human and Mouse Sera

An aliquot of 625 μL of a 250 μM solution of tested compound in PBS (pH 7.4) was added to 1.875 mL of mouse (Europa Bioproducts, EQSM-0100) or human serum (Sigma-Aldrich) prewarmed at 37 °C. Next, the solution was incubated at 37 °C for 4 h, taking aliquots of 250 μL at different times (0, 1, 2, 4, 8, 24, and 48 h). Each aliquot was quenched in 375 μL of cold ACN, vortexed, incubated for 10 min in ice and centrifuged at 39,000g for 10 min. Supernatants were then analyzed by HPLC-MS using SIM mode, and quantification was estimated by using the peak area integration normalized with an internal standard.

Stability Assays in Mouse and Human Liver Microsomes

Tested compound was incubated at 37 °C in 1 μM PBS with NADPH (final concentration of 2 mM) and MgCl₂ (final concentration of 5 mM). Metabolic reactions were initiated by the addition of a suspension of MLMs (male CD-1 mice pooled, Sigma-Aldrich) or HLMs (male human pooled, Sigma-Aldrich), at a final protein concentration of 1 mg/mL. The solutions were shaken in a vortex and kept at 37 °C in a water bath open to the air. Aliquots of 100 μL were quenched at time zero and at seven points ranging to 2 h (MLM) or 8 h (HLM) by pouring into 100 μL of ice-cold ACN. Quenched samples were centrifuged at 10,000g for 5 min, and the supernatants were filtered through a poly­(tetrafluoroethylene) membrane syringe filter (pore size of 0.2 μm, Albet Labscience). The relative loss of parent compound over the course of the incubation was monitored by HPLC-MS using SIM mode. Concentrations were quantified by measuring the area under the peak ([M + H]⁺) and converted to the percentage of remaining compound, using the time zero peak area value as 100%. The natural logarithm of the percentage remaining versus time data for each compound was fitted to linear regression, and the slope was used to calculate the degradation half-life time (t _1/2).

Parallel Artificial Membrane Permeability Assay (PAMPA)

Prior to use, the commercially available 96-well Corning Gentest precoated PAMPA plate system (Cultek S.L.U., Spain) was warmed to rt for 30 min. Then, 300 μL of 200 μM solution of tested compound in 2% DMSO in PBS were added into wells in the donor plate, and 200 μL of PBS were added into wells in the acceptor plate. The acceptor plate was placed on the donor plate by lowering the precoated PAMPA plate. The assembly was incubated at rt for 5 h, and then buffer samples were collected from each plate. The final concentrations of compound in both donor and acceptor wells were analyzed by HPLC-MS using SIM mode and quantification was estimated by using the peak area integration normalized with an internal standard. Permeability values of tested compounds and propranolol and metoprolol as reference compounds were calculated using the following formula: P (cm/s) = {−ln­[1 – C _A(t)/C _eq]}/[A × (1/V _D + 1/V _A) × t], where A = filter area (0.3 cm²), V _D = donor well volume (0.3 mL), V _A = acceptor well volume (0.2 mL), t = incubation time (s), C _A(t) = compound concentration (μM) in acceptor well at time t, C _D(t) = compound concentration (μM) in donor well at time t, and C _eq = [C _D(t) × V _D + C _A(t) × V _A]/(V _D + V _A).

hERG Assay

Interaction of the compounds with the hERG potassium channel was evaluated by Fundación Medina using the FluxOR potassium channel assay, using hERG-HEK293 cell line that stably expresses at passage 18. The assay was performed as outlined in the Invitrogen information sheet and performed on the FLIPR TETRA (Molecular Devices). Powerload concentrate and water-soluble probenecid were added in the first step, followed by FluxOR. Loading buffer was 165 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl, 10 mM Hepes and 10 mM glucose, adjusted to pH 7.2. Media were removed from the cell plates and 50 μL of loading buffer containing the FluxOR dye mix was applied to each for 60 min at rt, then removed manually. The cell plates were subsequently washed once with assay buffer, before adding the samples at 100 μM a final volume of 50 μL of assay buffer. Plates were incubated at rt for 30 min to allow equilibration of the test compounds. Stimulation buffer (Tl₂SO₄ + K₂SO₄) was prepared following the manufacturer instruction. The injection of stimulation buffer into the plates was performed on the FLIPR TETRA, and the kinetic data obtained for 8 points from time 0 to 120 s were analyzed using Genedata Screener. Positive control (20 μM astemizole) and negative control (1% DMSO) were introduced.

In Vivo Experiments

Animal procedures were approved by the Bioethics Committee of Santiago de Compostela University in compliance with the Principles of Laboratory Animal Care of national laws (license number 15012/2022/020).

Pharmacokinetics

Tested compound was administered (40 mg/kg, i.p.) and blood was collected by cardiac puncture at the selected time points postdose (n = 2 per time point). Blood was allowed to clot at rt for 20 min and centrifuged at 4 °C for 10 min at 10,000g. The supernatant was transferred to a clean polypropylene tube and stored at −80 °C until analysis. For analysis, a volume of cold ACN was added to the serum. The sample was incubated in an ice bath for 10 min and centrifuged at 4 °C for 10 min at 18,000g. The resulting organic layer was filtered through a poly­(tetrafluoroethylene) filter (0.2 μM, 13 mm diameter, Fisher Scientific) and 20 μL of the sample were analyzed by LC-MS/MS (UCM’s mass spectrometry facilities). Separation was performed using an Agilent Zorbax Rx-SIL column (C18, 5 μm 80 Å 250 × 4.6 mm), with a run time of 8 min and a flow of 0.4 mL/min (gradient: 3 min 5% to 35% B; 5 min 35% to 100% B; 8 min 5% B; phase A: water with formic acid 0.1%; phase B: ACN). The entire eluent was directly introduced to an electrospray ionization source operating in the positive ion mode in a Shimadzu LCMS8030 triple quadrupole mass spectrometer coupled to UHPLC with an oven temperature of 31.5 °C. The mass spectrometer ion optics were set in the multiple reaction monitoring mode and the transition selected for quantification was set in the molecular weight range. Concentrations were quantified by measuring the area under the peak (AUC) registered for the compound ([M + H]⁺) in each sample. The natural logarithm of the concentration versus time data for each compound was fitted to linear regression, and the slope was used to calculate the elimination rate constant (k _e).

Pulmonary Fibrosis Model

Male C57BL/6J 10 weeks old mice were intratracheally instilled with bleomycin (1 IU/kg, Mylan Pharmaceuticals) under anesthesia. After 2 weeks, animals were treated with the tested compound (i.p. at 40 mg/kg) or ABT-263 (AbbVie, p.o. at 50 mg/kg) daily for 1 week. At the end of the experiment, animals were sacrificed and lungs were removed for analysis.

Tissue Staining

SA-β-gal staining was performed following well-established methods. Briefly, whole mount lungs were fixed at rt in 2% formaldehyde/0.2% glutaraldehyde, washed, and incubated overnight at 37 °C with fresh SA-β-gal staining solution: 1 mg of 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal) per mL (Fisher Scientific), 40 mM citric acid/sodium phosphate (pH 5.6), 5 mM K₃Fe­[CN]₆, 5 mM K4Fe­[CN]₆, 150 mM NaCl, and 2 mM MgCl₂. Tissues were then embedded in paraffin, sectioned, and counterstained with Nuclear Fast Red.

Masson trichrome staining was performed by the Histopathology Unit at the Clinical University Hospital of Santiago de Compostela following standard protocols.

Stained tissue sections were visualized, and pictures were taken with AxioVert.A1Microscope (Zeiss). The images were analyzed with Zen Blue Edition software (Zeiss) and ImageJ (FIJI) for the quantification of the positive area for each marker.

Q-RT-PCR

To assess gene expression, total RNA was isolated from lung tissues using the NucleoSpin RNA Kit (Macherey-Nagel) as per the manufacturer’s guidelines and converted to cDNA through High-Capacity cDNA Reverse Transcription (Applied Biosystems). For quantification, we utilized the NZYSpeedy qPCR Green Master Mix reagent (2X), ROX (NZYTech), and the AriaMx Real-Time PCR systems thermocycler (Agilent Technologies). Each reaction included 33 ng of cDNA, oligonucleotides at a final concentration of 0.25 μM, 5 μL SYBR, and nuclease-free water, resulting in a final volume of 10 μL per reaction. GAPDH served as the housekeeping gene, and the gene expression levels were normalized to its expression. Triplicate analyses were conducted for each sample. The results were evaluated with the AriaMx 1.0 software (Agilent Technologies), and the primers (Table ) were sourced from Eurofins Genomics.

4. Primers for Q-RT-PCR.

Gapdh	5′-TCCATGACAACTTTGGCATCGTGG-3′
	5′-GTTGCTGTTGAAGTCACAGGAGAC-3′
Cdkn1a	5′-GTGGGTCTGACTCCAGCCC-3′
	5′-CCTTCTCGTGAGACGCTTAC-3′
Col1a1	5′- TTCTCCTGGCAAAGACGGACTCAA-3′
	5′- AGGAAGCTGAAGTCATAACCGCCA-3′
Ccl2	5′- CATCCACGTGTTGGCTCA-3′
	5′- GATCATCTTGCTGGTGAATGAGT-3′

Statistics

Statistical analysis was conducted using Prism Software (GraphPad, v.10.4.2). Data are presented as mean ± SD unless indicated otherwise. Group allocation occurred randomly. For in vitro data that are normally distributed with equal variance, we assessed statistical significance using a two-tailed unpaired Student’s t test. For in vivo data, the nonparametric Mann–Whitney test was used.

Glossary

Abbreviations Used

ACN

acetonitrile

ATR

attenuated total reflection

br

broad

CL

clearance

cpr

cyclopropane

DIPEA

N,N-diisopropylethylamine

DMEM

Dulbecco’s modified Eagle’s medium

dr

diastereoisomeric ratio

EDC

N-(3-(dimethylamino)­propyl)-N′-ethylcarbodiimide

FBS

fetal bovine serum

FDG

fluorescein-di-β-galactopyranoside

HLM

human liver microsomes

ind

indole

k _e

elimination rate constant

MLM

mouse liver microsomes

morp

morpholine

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

nd

not determined

ox

oxazole

P

permeability value

py

pyridine

quint

quintuplet

SA-β-gal

senescence-associated β-galactosidase

SASP

senescence-associated secretory phenotype

SEM

standard error of the mean

SIM

selected ion monitoring

thz

thiazole

UCM

Universidad Complutense de Madrid

V _D

distribution volume

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00412.

• Tables S1 and S2, Figures S1–S3, synthesis and characterization data of final compounds 2–24, 26–33, 85–89, and 97, and intermediates 36–61, 63–76, 78–84, 90–96, 99, 101, NMR, HPLC-MS, and HRMS spectra of final compounds 25 and 98 (PDF)

• Molecular formula strings (CSV)

The manuscript was written through contributions of all authors. H.V.-V., M.L.L-R., and B.B. designed the study, analyzed data, and wrote the original draft of the manuscript. I.A.-A., S.A., and A.S.-M. performed the synthesis and characterization of new compounds. I.A.-A. and B.M.-R. determined β-galactosidase activity. I.A.-A. and S.A. carried out ADME and pharmacokinetic assays. P.P.-R. and M.C. designed and conducted experiments in human lung cells and in the mouse pulmonary fibrosis model, and analyzed data. The manuscript was revised with contributions from P.P.-R., M.C., H.V.-V., and B.B.

This work was supported by grants PID2019–106279-RBI00, funded by MCIN/AEI/10.13039/501100011033, and PID2022–138797OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. The authors acknowledge technological support from NMR, mass spectrometry, and elemental analysis CAIs (Complutense University of Madrid). Work in the laboratory of M. Collado is funded by grants from MCINN/AEI/FEDER, EU (PID2021–125479OB-I00 and PID2024–159335OB-I00) and GAIN, Xunta de Galicia (IN607B2024/13). I.A.-A., S.A., and A.S.-M. thank the Ministerio de Ciencia e Innovación and Complutense University of Madrid for predoctoral fellowships. P.P.R. is grateful to Xunta de Galicia for the postdoctoral fellowship (IN606B-2023/001). Biorender was used to prepare the graphical abstract.

The authors declare no competing financial interest.